CEE 697z Organic Compounds in Water and Wastewater Cyanotoxins - - PowerPoint PPT Presentation

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Print version CEE 697z Organic Compounds in Water and Wastewater Cyanotoxins qPCR Method Prepared and presented by Kristie Stauch-White Lecture #29 CEE 697z - Lecture #29 qPCR Quantification of Microcystis during Lake Erie Blooms in 2003 and

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CEE 697z

Organic Compounds in Water and Wastewater

Cyanotoxins qPCR Method Prepared and presented by Kristie Stauch-White

CEE 697z - Lecture #29

Print version

Lecture #29

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LANDSAT 7 Image of Western Basin of Lake Erie near the mouth of the Maumee River

Maumee River

Rinta-Kanto, 2005

qPCR Quantification of Microcystis during Lake Erie Blooms in 2003 and 2004

CEE 697z - Lecture #29

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Anabaena Oscillatoria Agardhii

Microcystin Producers

CEE 697z - Lecture #29

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CEE 697z - Lecture #29

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Animal Bioassays
Immunoassays:
ELISA
PPIA
qPCR

CEE 697z - Lecture #29

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ELISA - Enzyme Linked Immunosorbent Assay

Lowest Detection Limit .05 ppb to .5 ppb

Direct Competitive ELISA using Polyclonal Antibody

Anti-Microcystin antibody attached to a high binding capacity microtiter plate
MCYST-LR is used as a standard
Microcystin-LR-peroxidase used to compete with MCYST-LR for the binding site of

the antibody on the microtiter plate.

Color developed inversely proportional

to MCYST concentration (lighter color = higher MCYST concentration)

http://www.jove.com/science-education/5061/the-elisa-method CEE 697z - Lecture #29

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PPIA

CEE 697z - Lecture #29

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MCYSTs and NODLNs – naturally potent inhibitors of protein serine

and threonin phosphatases (PP1 and PP2A)

Protein phosphatases dephosphorylate p-nitrophenylphosphate

(pNPP) – commonly used substrate for alkaline phosphatases

Colorimetric
Detection limit similar to ELISA (1 – 20 μg/L)
Advantage over ELISA – ability to detect bioactivity in MCYSTs and

NODLNs – therefore based on functional activity rather than structure

PPIA (Phosphatase Inhibition Assay) – How it works:

CEE 697z - Lecture #29

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mcyB 2959F mcyB 3278R TGGGAAGATGTTCTTCAGGTATCCAA AGAGTGGAAACAATATGATAAGCTAC 26 bp each, PCR product 60 – 1600 bp Wikipedia

Polymerase Chain Reaction - PCR

Primers:

60oC

CEE 697z - Lecture #29

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Rinta-Kanto, 2005

CEE 697z - Lecture #29

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*Toxin detected, but toxin-producing genes not detected at this site

* Neither toxin producing gene found at this site, but microcystin detected (see Table 3)

Rinta-Kanto, 2005

CEE 697z - Lecture #29

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Applied Biosystems

qPCR Fluorescence Emission

CEE 697z - Lecture #29

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Bio-Rad

P = I * En

P is PCR product amount I is initial quantity of DNA E is PCR efficiency N is number of cycles

CEE 697z - Lecture #29

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Eff = 10^(-1/slope) Eff% = ((10^(-1/slope))-1)*100%

CEE 697z - Lecture #29

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Rinta-Kanto, 2005

CEE 697z - Lecture #29

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CEE 697z - Lecture #29

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